JP6707072B2 - Nucleic acid for nucleic acid amplification - Google Patents

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority to US Provisional Patent Application No. 61/617562 filed March 29, 2012, the entire contents of which are incorporated herein by reference. Is.

Reference to Sequence Listing This application has been filed with the Sequence Listing in electronic form. The sequence listing has a size of 15,257 bytes, was created on March 13, 2013, and was last saved on March 13, 2013 PCT_SEQLIST_GENOM_115 WO. It is provided as a file titled txt. This information is incorporated herein by reference in its entirety.

Embodiments herein generally relate to internal control nucleic acids useful in monitoring nucleic acid amplification and/or extraction from a sample in a nucleic acid test (NAT).

Nucleic acid test (NAT) assays provide a powerful tool for rapid detection and/or quantification of target nucleic acids. Therefore, NAT assays are commonly used to detect the presence of organisms in a sample, eg, a patient sample in a clinical setting, a food sample, an environmental sample, and the like. NAT assays are also commonly used in diagnostic settings to detect genetic polymorphisms, gene repeats, insertions, deletions, etc., or changes in gene expression, eg, as an indication of a condition such as a disease or disorder. It

In many situations, it is desirable to have quantitative information regarding the amount of target nucleic acid sequences in a given sample. For example, a quantitative nucleic acid assay, eg, an amplification assay, detects the presence and/or amount of pathogen-specific target sequences present in a biological sample to determine whether the sample is infected with a pathogen, and It can be used to monitor the progress or severity of infection. Quantitative nucleic acid assays are used to monitor the condition of a cell or tissue by monitoring the amount of marker nucleic acid sequences present in the cell or tissue, or to specific DNA elements present in a sample, such as repeats or translocations. It may also be useful in quantifying the amount of factor.

Quantitative nucleic acid amplification reactions can be used to quantify the relative and/or absolute amount of target nucleic acid sequences present in a sample. Such methods have become highly advanced and sensitive so that a small number of copies of the target nucleic acid can be detected in the sample. Choose an appropriate internal control to avoid false positives, false negatives, overestimation of target or product, or underestimation of target or product due to the high sensitivity of the quantitative nucleic acid amplification reaction You must be very careful when doing this. In addition to considerations regarding specificity of internal controls (eg, internal control templates, primers, and/or probes), eg, hairpins, performing A/T at very low annealing temperatures, amplifying nucleic acids and/or probes. There are considerations regarding the unique characteristics of the control nucleic acid sequences, including the performance of G/C at very high annealing temperatures that do not make them applicable to hybridization. Furthermore, it may be desirable to use the same internal control template sequence, primer set, and probe in different multiplex reactions for different target sequences. However, many nucleic acid sequences are applicable for amplification and/or probe hybridization under a narrow set of reaction conditions and are not sufficiently robust to be used as internal controls under a wide variety of reaction conditions.

Thus, one of ordinary skill in the art will appreciate the complex nature of identifying internal and internal control sequences, robust combinations of primers, and probes to monitor nucleic acid amplification. Furthermore, one of skill in the art would have extensive experience in validating that polynucleotide template sequences, primer pairs, probes, or combinations thereof would serve as viable/sufficient internal controls for quantitative nucleic acid amplification. It will be appreciated that frequent validation is often performed.

Embodiments disclosed herein relate to improved compositions useful as internal controls in nucleic acid test (NAT) assays. Accordingly, provided herein are internal control reagents, kits containing internal control reagents, and methods of making and using them in nucleic acid testing assays. As described in more detail below, the internal control reagents disclosed herein exhibit highly advantageous properties, including extremely high sensitivity and reproducibility. Further, the internal control reagents disclosed herein are highly versatile and useful as controls in NAT assays to detect and/or quantify a wide array of different target sequences.

Thus, in some embodiments, polynucleotides are provided that can be used as templates in nucleic acid amplification reactions, including, but not limited to, quantitative and/or qualitative nucleic acid amplification reactions. In some embodiments, the polynucleotide can be used as an internal control template for quantitative nucleic acid test assays such as quantitative PCR. Exemplary internal control template polynucleotides include, and are essentially, variants of these, including the sequences of SEQ ID NOs: 1, 8, 9, 10, 11, 12, 13, 14, or 15, or subsequences thereof. Or consists of these.

In some embodiments, oligonucleotides are provided that can be used, for example, as primers for nucleic acid amplification to amplify standards and/or template control sequences used as internal controls. Oligonucleotides may include individual primers and/or primer pairs for nucleic acid amplification. In some embodiments, these primers include a control template sequence (eg, SEQ ID NOs: 1, 8, 9, 10, 11, 12, 13, 14, 15, and variants thereof including subsequences thereof). Can be used to amplify Exemplary primers include, consist essentially of, or consist of the sequences of SEQ ID NOs: 3, 4, 5, or 6, or variants thereof.

In some embodiments, oligonucleotides are provided that can be used as probes. In some embodiments, these probes are oligonucleotides capable of hybridizing to control sequence amplicons disclosed herein, eg, SEQ ID NOs: 1, 8, 9, 10, 11, 12, 13. , 14, 15, and variants thereof. An exemplary probe comprises, consists essentially of, or consists of the sequence of SEQ ID NO:2, or a variant thereof.

In some embodiments, kits are provided. The kit can include the polynucleotides and/or oligonucleotides described herein. In some embodiments, the kit can include a quantitative nucleic acid amplification internal control polynucleotide and/or oligonucleotide. The kit can include at least one of a polynucleotide template and a primer that specifically amplifies the template sequence. In some embodiments, the kit can include a pair of amplification primers that specifically amplify the template sequence. In some embodiments, the kit can optionally include a probe that specifically hybridizes to the polynucleotide template. The kits disclosed herein can optionally include a polymerase, reaction buffer, one or more dNTPs, or any combination thereof, and other reagents used in the amplification reaction.

Some embodiments provide a method of performing a NAT assay. In some embodiments, the method comprises the step of providing a polynucleotide template sequence comprising, consisting essentially of, or consisting of SEQ ID NO:1 and hybridizing the template sequence specifically to the template sequence. Contacting with at least one primer, eg, the primer of SEQ ID NO: 3, 4, 5, or 6. In some embodiments, the polynucleotide comprises a variant of SEQ ID NO:1. In some embodiments, the method comprises extending the primer, thus producing an amplicon of the template sequence. In some embodiments, the method comprises detecting the presence and/or amount of amplicon. In some embodiments, the detecting step comprises contacting the amplicon with a probe, the probe comprising a detectable moiety and binding to the amplicon. In some embodiments, the method comprises the step of determining the amount of probe specifically bound to the amplicon. In some embodiments, the determining step is performed in real time as the amplicon is generated. In some embodiments, the probe comprises a detectable moiety and the amount of signal from the detectable moiety is measured. In some embodiments, the accumulation of probe specifically hybridized or bound to the amplicon is measured as a function of time or cycle and an amplification curve is generated. In some embodiments, a series of reactions is performed, each reaction providing a different known amount of template sequence. In some embodiments, the method comprises generating a calibration curve from a series of amplification curves. In some embodiments, a calibration curve is used to determine the initial concentration of polynucleotide sequences in the amplification reaction. In some embodiments, the amplification reaction comprises a multiplex amplification reaction.

FIG. 8 illustrates a vector map of a modified PUC119 vector having the sequence of SEQ ID NO:8. Sequence of modified PUC119 vector containing an insert containing an exemplary internal control template sequence ("DrosScaff2"; SEQ ID NO:8) (Fig. 2A shows positions 1-1800 of the sequence, Fig. 2B shows 1801 of the sequence). ~position 3350), and the relative position of the primer comprising SEQ ID NO:3 and SEQ ID NO:4, and the probe comprising SEQ ID NO:2. As noted by the diamond symbol, the "t" residue at position 1668 of this sequence differs from the "C" residue found at the corresponding position in SEQ ID NO: 15 ("DrosScaff1"). FIG. 8 illustrates a calibration curve derived from a real-time amplification reaction of 5 copies of template, 50 copies of template, and an internal control template containing 500 copies of SEQ ID NO: 8 using amplification primer pair 5. Cq was determined by performing 4 replicates for each starting amount of 5 copies, 50 copies and 500 copies of template. The calibration curve represents Cq vs. log starting amount of template. FIG. 3B shows an amplification curve (fluorescence vs. cycle) of a series of amplification reactions used to generate the calibration curve shown in FIG. 3A. Amplification curve (fluorescence vs cycle) of a series of amplification reactions where the initial template concentration was 200, 50, 25, or 5 copies of the linearized plasmid containing SEQ ID NO:8 and primer pair 5 was used as the amplification primer. FIG. A fluorescent probe containing the sequence SEQ ID NO: 2 was used in the reaction. Amplification curves of a series of real-time amplification reactions (fluorescence vs. cycle) where the initial template concentration was 200, 50, 25, or 5 copies of the linearized plasmid containing SEQ ID NO:8 and primer pair 1 was used as the amplification primer FIG. A fluorescent probe containing the sequence SEQ ID NO: 2 was used in the reaction. Amplification curves for a series of real-time amplification reactions (fluorescence vs. cycle) where the initial template concentration was 200, 50, 25, or 5 copies of the linearized plasmid containing SEQ ID NO:8 and primer pair 5 was used as the amplification primer FIG. A fluorescent probe containing the sequence SEQ ID NO: 2 was used in the reaction. Amplification curves for a series of real-time amplification reactions (fluorescence vs. cycle) where the initial template concentration was 200, 50, 25, or 5 copies of the linearized plasmid containing SEQ ID NO:8 and primer pair 5 was used as the amplification primer. FIG. A fluorescent probe containing the sequence SEQ ID NO: 2 was used in the reaction. FIG. 9 illustrates PCR performance (PCR efficiency, R2, and slope), and sensitivity, detecting as few as 5 copies/reaction of a linearized plasmid having the sequence of SEQ ID NO: 15 (“DrosScaff1” plasmid).

It should be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not intended to limit the scope of the present teachings. In this application, the use of the singular includes the plural unless specifically stated otherwise. Also, "comprises," "includes," and "includes," or modifications of these roots, such as, but not limited to, "comprises," "contained," and " The use of "including" is not intended to be limiting. The use of "or" means "and/or" unless stated otherwise. The term “and/or” means that the terms before and after can be adopted together or separately. For purposes of illustration and not limitation, “X and/or Y” may mean “X” or “Y”, or “X and Y”.

Whenever a range of values is provided herein, the range is meant to include the starting and ending values and any value or range of values there between, unless expressly stated otherwise. To do. For example, "0.2 to 0.5" means 0.2, 0.3, 0.4, 0.5; a range therebetween, for example, 0.2 to 0.3, 0.3 to 0.4. , 0.2 to 0.4, etc.; increments in between, eg, 0.25, 0.35, 0.225, 0.335, 0.49, etc.; increment range in between, such as 0.26 to 0.39. And so on.

The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All references and similar materials cited in this application, including but not limited to patents, patent applications, articles, books, papers, and Internet web pages, regardless of the format of such references and similar materials. , And these are expressly incorporated by reference in their entirety for any purpose. To the extent that one or more of the incorporated documents and similar material defines or uses a term in a manner that is inconsistent with the definition of that term in this application, the present application controls. Although the present teachings are described in connection with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents as will be appreciated by those skilled in the art.

Various embodiments of the present disclosure describe compositions and kits for use in nucleic acid testing (NAT) assays, and methods of using these. Thus, some embodiments provide nucleic acid sequences for use in NAT assays, eg, amplification assays. Those skilled in the art will appreciate that for any nucleic acid sequence, the reverse complement can be readily obtained and the disclosure of one nucleic acid sequence also discloses the reverse complement of that sequence. One of ordinary skill in the art will appreciate that subsequences of the nucleic acid sequences disclosed herein can be readily obtained.

The nucleic acids provided herein can be in various forms. For example, in some embodiments, the nucleic acid is dissolved in a solution, eg, a buffer (alone or in combination with various other nucleic acids). In some embodiments, nucleic acids are provided as salts, alone or in combination with other isolated nucleic acids. In some embodiments, nucleic acids are provided in lyophilized form that can be reconstituted. For example, in some embodiments, the isolated nucleic acids disclosed herein can be provided in a lyophilized pellet alone or in a lyophilized pellet containing other isolated nucleic acids. In some embodiments, nucleic acids are provided attached to a solid material, eg, beads, membranes, etc. In some embodiments, the nucleic acid is provided in a host cell, eg, a cell line that carries a plasmid or a cell line that carries a stably integrated sequence.

Control Sequences Some embodiments disclosed herein provide control nucleic acid sequences for NAT assays. In some embodiments, the control sequence can be used as an external control or standard that is processed in parallel with the test sample. In some embodiments, a control sequence can be used as an internal control and combined with the test sample prior to processing.

The term "nucleic acid" refers to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and N- or C-glycosides of purine or pyrimidine bases. Any other type of nucleotide, as well as other polymers containing non-nucleotide backbones, such as polyamides (eg, peptide nucleic acid (PNA)), and polymorpholino (NEUGENE™ polymers as Anti-Virals, Inc., Corvallis. , Oreg.), and other synthetic sequence-specific nucleic acid polymers, where the polymer is a nucleic acid in a configuration that allows base pairing and stacking, such as those found in DNA and RNA. One of ordinary skill in the art will appreciate that it contains a base.

The term nucleotide and polynucleotide include, for example, 3'-deoxy-2',5'-DNA, oligodeoxyribonucleotide N3'→P5' phosphoramidate, 2'-O-alkyl substituted RNA, double-stranded DNA and single strand. Includes stranded DNA, as well as double stranded and single stranded RNA, DNA:RNA hybrids, and hybrids between PNA and DNA or RNA. The term refers to known types of modifications, such as labels known in the art, methylation, "caps", substitution of naturally occurring nucleotides with one or more analogs, internucleotide modifications, such as , Uncharged linkages (eg, methyl phosphonate, phosphotriester, phosphoramidate, carbamate, etc.), negatively charged linkages (eg, phosphorothioate, phosphorodithioate, etc.), and positively charged linkages (eg, amino. Those having alkylphosphoramidates, aminoalkylphosphotriesters), for example, those containing pendant moieties such as proteins (including nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (eg , Acridine, psoralen, etc.), those containing chelators (eg, metals, radioactive metals, boron, metal oxides, etc.), those containing alkylating agents, those having modified linkages (eg, alpha anomers). Nucleic acid) and the like, as well as unmodified forms of polynucleotides or oligonucleotides.

It is understood herein that the terms "nucleoside" and "nucleotide" will include moieties that contain not only the known purine and pyrimidine bases, but also other modified heterocyclic bases. Let's do it. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles. Modified nucleosides or nucleotides will also include modifications on the sugar moiety, for example, one or more of the hydroxyl groups have been replaced with halogens, aliphatic groups, or functionalized as ethers, amines, etc. There is. Other modifications to the nucleotides or polynucleotides include rearrangement, addition, substitution of functional groups on purine or pyrimidine bases that form hydrogen bonds to their respective complementary pyrimidines or purines, such as isoguanine, isocysteine, etc. , Or otherwise with changes. In some embodiments, the oligonucleotide and/or probe comprises at least 1, 2, 3, or 4 modified nucleotides.

In some embodiments, the nucleic acids disclosed herein comprise one or more universal bases. As used herein, the term "universal base" refers to a nucleotide analog capable of hybridizing to more than one nucleotide selected from A, T, C, and G. In some embodiments, the universal base can be selected from the group consisting of deoxyinosine, 3-nitropyrrole, 4-nitroindole, 6-nitroindole, 5-nitroindole.

In some embodiments, the control nucleic acid sequence disclosed herein is a target control sequence ("TCS") that is a template for nucleic acid amplification reactions. A partial sequence of the TCS disclosed herein can be amplified by PCR or other amplification methods discussed in further detail herein so that the entire TCS disclosed herein can be amplified. One of ordinary skill in the art will appreciate that this is possible. In some embodiments, TCS is provided as a control in a multiplex assay in which a known amount of TCS and at least one target nucleic acid to be quantified are each amplified in the same reaction mixture. In some embodiments, a known amount of TCS is amplified in the first reaction mixture, while the target nucleic acid to be quantified is amplified in the second reaction mixture.

In some embodiments, the TCS consists of, consists essentially of, or comprises the sequence of SEQ ID NO:1, or variants thereof. In some embodiments, TCS is provided in a vector, eg, a plasmid. Thus, some embodiments consist of, consist of, or consist of, a polynucleotide sequence that consists of, consists essentially of, or comprises SEQ ID NO:1 in a plasmid, eg, SEQ ID NO:8. Provide an array. In embodiments where the TCS is provided in a plasmid, the plasmid may be linearized, or, alternatively, the plasmid may be non-linearized (eg, supercoiled or unrolled circular). In form). In some embodiments, the template sequence comprises a polynucleotide comprising at least one of the sequences of SEQ ID NOs: 9, 10, 11, 12, or 13, or variants thereof. "Variants" of the sequences disclosed herein are discussed below.

In some embodiments, TCS is, Drosophila spp., For example, Drosophila melanogaster, Drosophila simulans, Drosophila sechellia, Drosophila yakuba, Drosophila erecta, Drosophila ficusphila, Drosophila eugracilis, Drosophila biarmipes, Drosophila takahashii, Drosophila elegans, Drosophila rhopaloa, Drosophila kikkawai , Drosophila ananasae, Drosophila bispectinata, Drosophila pseudoobscura, Drosophila persimilis, or a genomic fragment of an organism containing Drosophila willistoni, or a modified fragment thereof, or a genomic fragment of an organism containing Drosophila willistoni. Preferably, the TCS sequence comprises, consists of, or consists essentially of SEQ ID NO:1 or SEQ ID NO:14. For example, SEQ ID NO: 14 generally corresponds to a sequence found in the genome of Drosophila melanogaster (GenBank: AC246436; nucleotides 35779 to 35978), SEQ ID NO: 1 comprises a modification to SEQ ID NO: 14, ie, of SEQ ID NO:14. The "C residue" at position 93 was changed to "T" at the same position in SEQ ID NO:1. This modification was also found in the plasmid of SEQ ID NO:8 and is annotated with a diamond symbol in Figure 2.

Oligonucleotides In some embodiments, oligonucleotides, eg, primers and/or probes, are provided. As used herein, the terms "primer" and "probe" include, but are not limited to, oligonucleotides. Preferably, the oligonucleotide primers and/or probes disclosed herein can be between 8 and 45 nucleotides in length. For example, the primers and/or probes are at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 in length. , 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or more nucleotides. Primers and/or probes can be provided in any suitable form, including, for example, solid support-bound, liquid, and lyophilized. The primer and probe sequences disclosed herein can be modified to contain additional nucleotides at the 5'or 3'ends, or both. However, one of ordinary skill in the art will appreciate that the additional bases at the 3'end of the amplification primer (not necessarily the probe) will generally be complementary to the template sequence. The primer and probe sequences disclosed herein can also be modified to remove nucleotides at the 5'or 3'ends. Those skilled in the art will appreciate that in order to function for amplification, the primer or probe will be of the minimum length and annealing temperature disclosed herein.

Oligonucleotide primers and probes can bind to these targets at an annealing temperature that is below the melting temperature ( _Tm ). As used herein, " _Tm " and "melting temperature" are interchangeable terms that refer to the temperature at which 50% of a population of double-stranded polynucleotide molecules dissociates into single strands. The formula for calculating the T _m of a polynucleotide is well known in the art. For example, T _m can be calculated by the following formula: T _m =69.3+0.41×(G+C)%−6-50/L, where L is the length of the probe in nucleotides. That's it. The T _{m of the} hybrid polynucleotide was also taken from the hybridization assay in 1 M salt and is the formula commonly used to calculate the T _m of PCR primers, ie [(number of A+T)×2° C.+(G+C). Number)×4° C.] can be used for the estimation. For example, C.I. R. See Newton et al., PCR, 2nd Edition, Springer-Verlag (New York: 1997), page 24. Other more sophisticated calculations exist in the art that take structural and sequence properties into account to calculate the T _m . The melting temperature of an oligonucleotide can depend on the complementarity between the oligonucleotide primer or probe and the binding sequence, and salt conditions. In some embodiments, the oligonucleotide primers or probes provided herein are less than about 90° C. in 50 mM KCl, 10 mM Tris-HCl buffer, eg, any two of the listed values. About 89°C, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, including the range between , 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 50, 49, 48, 47, 46, 45, 44, 43. has 42,41,40,39 ° C., or less _{T m.} As discussed in further detail below, in some embodiments, the primers disclosed herein are provided as amplification primer pairs that include, for example, a forward primer and a reverse primer. Preferably, the forward and reverse primers do not differ by more than 10°C, eg less than 10°C, less than 9°C, less than 8°C, less than 7°C, less than 6°C, less than 5°C, less than 4°C, 3°C. Having a T _m different by less than, less than 2°C, or less than 1°C.

The primer and probe sequences can be modified by having nucleotide substitutions (as compared to the target sequence) within the oligonucleotide sequence, provided that the oligonucleotide is sufficient to specifically hybridize to the target nucleic acid sequence. Contains a complementary nature. In this way, at least 1, 2, 3, 4, or up to about 5 nucleotides can be replaced. As used herein, the term "complementary" refers to sequence complementarity between regions of two polynucleotide chains or between two regions of the same polynucleotide chain. When the two regions are arranged in antiparallel fashion, the first region of the polynucleotide is the same or if at least one nucleotide of the first region is capable of base pairing with the base of the second region. It is complementary to a second region of a different polynucleotide. Therefore, it is not required that the two complementary polynucleotides base pair at every nucleotide position. "Perfectly complementary" refers to a first polynucleotide that is 100%, or "completely," complementary to a second polynucleotide and thus forms base pairs at all nucleotide positions. "Partially complementary" is also not 100% complementary (eg, 90%, or 80%, or 70% complementary) and contains a first nucleotide containing a mismatch at one or more nucleotide positions. Refers to a polynucleotide. In some embodiments, the oligonucleotide comprises universal bases.

The term "hybridization" is used herein with reference to the pairing of complementary (including partially complementary) polynucleotide strands. The strength of hybridization and hybridization (ie, the strength of association between polynucleotide strands) depends on the degree of complementarity between polynucleotides, the concentration of salts, the melting temperature of the hybrid formed, and the presence of other components. The art, including the stringency of the conditions involved, is influenced by conditions such as (eg, the presence or absence of polyethylene glycol), the molar concentration of the hybridizing strand, and the G:C content of the polynucleotide strand. Impacted by many factors known in the art. In some embodiments, the primer, the T _m of one primer in the set is designed to be within 2 ℃ in T _m of the other primers in the set. Extensive guides to nucleic acid hybridization can be found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with 95%, Nucleic Acids. It can be found in Protocols in Molecular Biology, Chapter 2, (Greene Publishing and Wiley-Interscience, New York). See Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd edition, Cold Spring Harbor Laboratory Press, Plainview, New York). As further discussed herein, the term "specific hybridization" or "specifically hybridize" refers to the target sequence under conditions commonly used for nucleic acid amplification, and not to unrelated sequences. , Refers to hybridization of a polynucleotide, eg, an oligonucleotide primer or probe, to a sequence to be quantified, a positive control target nucleic acid sequence, etc. in a sample.

In some embodiments, the primer and/or probe comprises an oligonucleotide that hybridizes to the target nucleic acid sequence over the entire length of the oligonucleotide sequence. Such sequences can be referred to as "fully complementary" to each other. When an oligonucleotide is referred to as "substantially complementary" to a nucleic acid sequence herein, the two sequences may be perfectly complementary, or they may form a mismatch upon hybridization. , May retain the ability to hybridize under stringent or standard PCR conditions, discussed below. As used herein, the term “substantially complementary” refers to the complementarity between two nucleic acids, eg, complementary regions of an oligonucleotide and a target sequence. Complementarity need not be perfect and there can be any number of base pair mismatches between two nucleic acids. However, if the number of mismatches is so great that no hybridization can occur even under the most stringent hybridization conditions, the sequences are not substantially complementary sequences. When two sequences are referred to herein as "substantially complementary," it means that the sequences are sufficiently complementary to each other to hybridize under the selected reaction conditions. The relationship between sufficient nucleic acid complementarity to achieve specificity and hybridization stringency is well known in the art and is further described below with reference to sequence identity, melting temperature, and hybridization conditions. Has been done. Thus, substantially complementary sequences can be used in any of the detection methods disclosed herein. Such probes can be, for example, perfectly complementary, or as long as the hybridization conditions are sufficient to allow, for example, discrimination between target and non-target sequences, from one to many. Mismatches can be included. Thus, a substantially complementary sequence is 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, compared to a reference sequence. A sequence can be referred to as having a range of percent identity of 84, 83, 82, 81, 80, 75, 70, or less, or any number in between. For example, an oligonucleotide disclosed herein may contain 1, 2, 3, 4, 5, or more mismatches and/or degenerate bases as compared to the target sequence to which the oligonucleotide hybridizes. Provided that the oligonucleotide is capable of specifically hybridizing to the target sequence under standard nucleic acid amplification conditions, for example.

The primers described herein can be prepared using techniques known in the art, including, but not limited to, cloning and digestion of appropriate sequences, and direct chemical synthesis. Chemical synthesis methods that can be used to make the primers described herein include, but are not limited to, the phosphotriester method described by Narang et al. (1979) Methods in Enzymology, 68:90, Brown et al. 1979) Methods in Enzymology, 68:109, the phosphodiester method, Beaucage et al. (1981) Tetrahedron Letters, 22:1859, and the diethylphosphoramidate method, and U.S. Pat. No. 4,458,066. There is a solid support method. The use of automated oligonucleotide synthesizers to prepare the synthetic oligonucleotide primers described herein is also contemplated herein. Furthermore, if desired, primers can be labeled using techniques known in the art and described below.

Preferably, the oligonucleotides disclosed herein are fully or substantially complementary to a target sequence or target polynucleotide, eg TCS. As used herein, the terms "target polynucleotide" and "target nucleic acid" refer to a polynucleotide whose presence is determined within a reaction, eg, an internal control sequence, and/or a sample sequence to be measured.

Accordingly, provided herein is a primer that comprises, consists essentially of, or consists of one sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, or a variant thereof.

Primer Sets In some embodiments, a set of amplification primers is provided. The set of amplification primers may be one or more, eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more primer pairs. Can be included. As used herein, the term “primer pair” can refer to two primers that individually hybridize to a target nucleic acid, eg, opposite strands of a quantitative PCR internal control sequence, where each primer is, eg, In PCR, it can be extended at its 3'end to form a target amplification product. A primer pair can include a forward primer and a reverse primer.

In some embodiments, the compositions and methods disclosed herein include a primer pair that includes at least one set of amplification primers that hybridize to and amplify a target control sequence. A first oligonucleotide comprising, consisting essentially of, or consisting of the sequence SEQ ID NO:3, and a second oligonucleotide comprising, consisting essentially of, or consisting of the sequence SEQ ID NO:4, 3 is an exemplary primer pair (hereinafter “primer pair 5”) useful in connection with the embodiments disclosed herein. A first oligonucleotide comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO:5, and a second oligonucleotide comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO:6. Another exemplary primer pair (hereinafter “primer pair 1”) useful in connection with the embodiments disclosed herein. The characteristics of primer pair 1 and primer pair 2 are listed in Table 1.

In some embodiments, the primer pair comprises a sequence of SEQ ID NO:3 or SEQ ID NO:5, or a first primer comprising a variant of SEQ ID NO:3 or SEQ ID NO:5, and a sequence of SEQ ID NO:4 or SEQ ID NO:6, or A second primer comprising a variant of SEQ ID NO:4 or SEQ ID NO:6.

In some embodiments, the primer pair is used to amplify a template containing a target control sequence (TCS) amplicon, such as the sequence of SEQ ID NO:1, the sequence of SEQ ID NO:8, or variants thereof. You can In some embodiments, primer pair 1 or primer pair 5 is used to amplify the sequence of SEQ ID NO: 1 or SEQ ID NO: 8 (eg, template control sequence). In some embodiments, therefore primer pair 5 produces the amplicon shown in SEQ ID NO:9. In some embodiments, primer pair 1 or primer pair 5 amplifies a variant comprising SEQ ID NO: 1, eg, a template comprising the sequence of SEQ ID NO: 10, 11, 12 or 13. In some embodiments, primer pair 1 or primer pair 5 amplifies a template that includes Drosophila genomic DNA, or genomic fragments.

Probes In some embodiments, sequence-specific probes are provided. Probes include, but are not limited to, the oligonucleotides described herein. In some embodiments, the sequence-specific probes disclosed herein specifically hybridize to a target sequence such as TCS. In some embodiments, the sequence-specific probe specifically hybridizes to and is completely or substantially complementary to a nucleotide sequence flanked by the binding sites of the forward and reverse primers disclosed herein. Is. In some embodiments, the sequence-specific probe is at least 5,6,7,8,9,10,11,12,13,14,15,16,17 of SEQ ID NO:3,4,5, or 6. , 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides, such that the sequence-specific probe overlaps the binding site of the amplification primer disclosed herein.

Various types of detectable moieties for detecting amplification products have been described. One class of detectable moieties are intercalating agents that bind non-specifically to double-stranded nucleic acids. The intercalating agent has a relatively low fluorescence when unbound and a relatively high fluorescence when bound to double-stranded nucleic acid. Therefore, the intercalating agent can be used to monitor the accumulation of double-stranded nucleic acid during the nucleic acid amplification reaction. Examples of such non-specific dyes include intercalating agents such as SYBRGreen I (Molecular Probe), propidium iodide, ethidium bromide. Another type of detectable moiety uses derivatives of sequence-specific nucleic acid probes. For example, an oligonucleotide probe labeled with one or more dyes such that it will produce a detectable change in fluorescence when hybridized to a template nucleic acid. Sequence-specific probes can provide a more accurate measurement of amplification, although non-specific dyes may be desirable for some applications. One configuration of a sequence-specific probe can include one end of the probe tethered to the fluorophore and the other end of the probe tethered to the quencher. When the probe is unhybridized, it can maintain a stem-loop configuration where the fluorophore is quenched by the quencher, thus preventing the fluorophore from fluorescing. When the probe is hybridizing to the template nucleic acid sequence, it is linearized, keeping the fluorophore away from the quencher and thus allowing the fluorophore to fluoresce. Another configuration of sequence-specific probes can include a first probe tethered to the first fluorophore of the FRET pair and a second probe tethered to the second fluorophore of the FRET pair. The first and second probes are amplifiers that are within close enough proximity to allow energy transfer by FRET when the first and second probes are hybridized to the same amplicon. It can be arranged to hybridize to the sequence of the recon.

In some embodiments, the sequence-specific probe comprises an oligonucleotide disclosed herein conjugated to a fluorophore. In some embodiments, the probe is conjugated to more than one fluorophore. Examples of fluorophores include xanthene dyes such as fluorescein and rhodamine dyes such as fluorescein isothiocyanate (FITC), 2-[(ethylamino)-3-(ethylimino)-2-7-dimethyl-3H-xanthene- 9-yl]benzoic acid ethyl ester monohydrochloride (R6G) (which emits response radiation in the wavelength range of about 500-560 nm), 1,1,3,3,3',3'-hexamethyl Indodicarbocyanine iodide (HIDC) (which emits responsive radiation at wavelengths in the range of about 600-660 nm), 6-carboxyfluorescein (commonly known by the abbreviations FAM and F), 6-carboxy-2',4',7. ',4,7-Hexachlorofluorescein (HEX), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE or J), N,N,N',N'-tetramethyl -6-carboxyrhodamine (TAMRA or T), 6-carboxy-X-rhodamine (ROX or R), 5-carboxyrhodamine-6G (R6G5 or G5), 6-carboxyrhodamine-6G (R6G6 or G6), and rhodamine 110 and the like; cyanine dyes such as Cy3, Cy5, and Cy7 dyes; coumarins such as umbelliferone; benzimide dyes such as Hoechst33258; phenanthridine dyes such as Texas Red; ethidium dyes; acridine dyes; carbazole dyes; Phenoxazine dyes; Porphyrin dyes; Polymethine dyes, for example, cyanine dyes such as Cy3 (which emits response radiation in a wavelength range of about 540 to 580 nm), Cy5 (which emits response radiation in a wavelength range of about 640 to 680 nm), and the like. There are BODIPY dyes, as well as quinoline dyes. Specific fluorophores of interest include pyrene, coumarin, diethylaminocoumarin, FAM, fluorescein chlorotriazinyl, fluorescein, R110, eosin, JOE, R6G, HIDC, tetramethylrhodamine, TAMRA, lyssamin, ROX, naphthofluorescein, Such as Texas Red, naphthofluorescein, Cy3, and Cy5.

In some embodiments, the probe is conjugated to the quencher. The quencher is capable of absorbing electromagnetic radiation and dissipating it as heat, so it remains dark. Exemplary quenchers include Dabcyl, NFQ, such as BHQ-1 or BHQ-2 (Biosearch), IOWA BLACK FQ (IDT), and IOWA BLACK RQ (IDT). In some embodiments, the quencher is selected to pair with the fluorophore to absorb the electromagnetic radiation emitted by the fluorophore. Fluorophore/quencher pairs useful in the compositions and methods disclosed herein are well known in the art and can be found at the worldwide website molecular-beacons. org/download/marras, mmb06% 28335% 293. S.P.D. Marras, "Selection of Fluorophore and Quencher Pairs for Fluorescent Nucleic Acid Hybridization Probes", and may be described, for example.

In some embodiments, the fluorophore is attached to the first end of the probe and the quencher is attached to the second end of the probe. The attachment can include covalent bonds and can optionally include at least one linker molecule located between the probe and the fluorophore or quencher. In some embodiments, the fluorophore is attached to the 5'end of the probe and the quencher is attached to the 3'end of the probe. In some embodiments, the fluorophore is attached to the 3'end of the probe and the quencher is attached to the 5'end of the probe. Examples of probes that can be used in quantitative nucleic acid amplification include molecular beacons, SCORPIONS™ probes (Sigma), and TAQMAN™ probes (Life Technologies).

Some embodiments disclosed herein provide a probe that specifically hybridizes to the template control sequence of SEQ ID NO:1 or SEQ ID NO:8, or an amplicon of the template control sequence, or a subsequence thereof. Accordingly, some embodiments disclosed herein provide a probe that hybridizes to an amplicon from the amplification of a template comprising SEQ ID NO: 1 or SEQ ID NO: 8 by one of primer pair 1 or primer pair 5. .. In some embodiments, the probe measures amplification of a variant of SEQ ID NO: 1 or a subsequence thereof by hybridizing to at least one amplicon of SEQ ID NO: 8, 9, 10, 11, 12, or 13. To do. Thus, in some embodiments, an oligonucleotide probe is provided that comprises, consists essentially of, or consists of the sequence of SEQ ID NO:2 or 16, or a variant thereof. In some embodiments, the probe comprises a fluorophore and/or quencher as described herein. In a preferred embodiment, the probe has a fluorophore 6-carboxy-X-rhodamine ("ROX" or "R") attached to the 5'end of the probe and is a quencher IOWA BLACK Black-hole Quencher®. )2(IDT) ("BHQ") can include SEQ ID NO:2 attached to the 3'end of the probe (e.g., 5'-(ROX)-TGA TGC CTC TTC ACA TTG CTC CAC CTT TCC TCT. -BHQ-2-3').

In some embodiments, more than one probe is provided. In some embodiments, a first probe is provided and a second probe is provided. The first probe can be attached to the first FRET fluorophore. The second probe can be attached to the second FRET fluorophore. Each of the first probe and the second probe can be arranged to hybridize to the sequence of the amplicon to be quantified. In some embodiments, the first probe is positioned to hybridize to the first partial sequence of the amplicon and the second probe is hybridized to the second partial sequence of the amplicon. Will be placed. In some embodiments, the first subsequence is located 5'of the second subsequence and is a base between the 3'end of the first subsequence and the 5'end of the second subsequence. Is about 10 bases or less, for example 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 bases. In some embodiments, the first probe and the second probe each hybridize to a subsequence of SEQ ID NO:1, or a sequence that is a variant thereof. In some embodiments, the first probe comprises the sequence of SEQ ID NO:2, or a variant or subsequence of SEQ ID NO:2, and the second probe is as described herein.

Vectors In some embodiments, the nucleic acid sequences provided herein (eg, TCS) are in a vector, eg, a plasmid or virus. Vectors are well known in the art and the skilled artisan will understand that many vectors may be used to provide the nucleic acid sequences. Moreover, there are many variants of the vector and additional vectors can be produced by those of skill in the art. The vector is modified by site-directed mutagenesis, random mutagenesis, or by one or more cloning steps to provide at least one nucleic acid sequence of the vector, eg, multiple cloning sites, antibiotic resistance gene, Markers that facilitate visualization of the origin or replication, or cells carrying the vector, such as genes encoding beta-galactosidase, luciferase, or green fluorescent protein, can be added, removed, and/or replaced.

In some embodiments, the vector contains a nucleic acid amplification template sequence. The nucleic acid amplification template sequence is, for example, by restriction digestion to remove the nucleic acid amplification template sequence from the first vector, and ligation to insert the nucleic acid amplification template sequence into the second vector, or the nucleic acid of the nucleic acid amplification template sequence. Amplification, eg, PCR amplification, and ligation of nucleic acid amplification template sequences into a second vector can be transferred to different vectors.

As an example, the plasmid derived from pUC119 is shown in SEQ ID NO:7. In some embodiments, a plasmid that does not contain an internal control template sequence (eg, TCS), such as the plasmid of SEQ ID NO:7, can be used as a negative control in the NAT assay. The sequence of the pUC119-derived plasmid containing the PCR template sequence of SEQ ID NO:1 is provided in SEQ ID NO:8. FIG. 1 illustrates a map of a plasmid containing the sequence of SEQ ID NO:8.

Variants that the listed nucleic acid sequence variants can be generated using techniques known in the art, for example, by random mutagenesis, site-directed mutagenesis, or chemical synthesis of the desired variant, Those skilled in the art will understand. In some embodiments, variants of the recited sequences are provided, where each variant has a sequence that differs from the reference sequence by at least one nucleotide. In some embodiments, variant nucleic acids are at least about 70%, 71%, 72%, 73%, including the reference sequence and at least about 70%, eg, including the range between any two of the recited values. , 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, 99 It comprises substantially complementary sequences with nt-nt identities of 9.9%, or 99.99%.

In some embodiments, the variant has substantially equivalent or similar function to the reference sequence. For example, for reference sequences that have primer and/or probe binding sites, many variants in the nucleic acid outside the primer and/or probe binding side do not affect primer binding and subsequent amplification or probe hybridization.

In some embodiments, the variant of the reference template is amplified with the same primers as the reference template. In some embodiments, the variant of the reference probe hybridization site hybridizes to the same probe as the reference sequence. In some embodiments, variants of TCS are amplified with the same primers and hybridized with the same primers as TCS. In some embodiments, variants of TCS are provided, and one or more corresponding variant primers are provided to anneal to the variant TCS. In some embodiments, variants of the probe binding site are provided and one or more corresponding variant probes are provided to hybridize to the variant probe binding site. In some embodiments, a variant of the quantitative nucleic acid amplification internal control sequence is provided and the corresponding variant primer and/or probe is provided to anneal to the variant internal control sequence.

In some embodiments, the variant results in a partial mismatch in the primer and/or probe hybridization site, but still allows binding of the primer or probe. Variants can be in probes and/or primers. Alternatively, the variant may be in the sequence to which the probe and/or primer binds. In some embodiments, the primer or probe variant results in a mismatch of 5 nucleic acids or less, eg, 5, 4, 3, 2, or 1 nucleotide. In some embodiments, even if a mismatch is present, the primer will anneal to the binding site and will function for PCR amplification unless the mismatch is within the first two nucleotides at the 3'end of the primer. it can. In some embodiments, the probe is capable of hybridizing to the target sequence, regardless of the location of the mismatch, even if a mismatch is present.

In some embodiments, the variant PCR primer or probe binds to the target sequence at the annealing temperatures described herein and is thus functional for amplification and/or detection of nucleic acid sequences.

In some embodiments, SEQ ID NO: 1 and at least about 85%, such as at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, Variants having 96%, 97%, 98%, 99% or more sequence identity are provided. SEQ ID NO: 1 represents the isolated sequence of Drosophila melanogaster. When the primers of SEQ ID NO:3 and SEQ ID NO:4 are used to amplify SEQ ID NO:1, the sequence of SEQ ID NO:9 is produced. In some embodiments, a primer pair comprising a forward primer comprising the sequence of SEQ ID NO:3 or SEQ ID NO:5 and a reverse primer comprising the sequence of SEQ ID NO:4 or SEQ ID NO:6 amplifies the genomic DNA of Drosophila melanogaster. Used to do. Due to the presence of repeats in this genomic DNA, sequences containing SEQ ID NOs: 8, 9, 10, or 11, or subsequences thereof, can be generated by the primer pairs described herein. In some embodiments, the sequence comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more nucleotide variants of the sequence of SEQ ID NO:1, but this sequence Can be amplified by a primer pair comprising one of the primers comprising the sequence of SEQ ID NO:3 or SEQ ID NO:5 and one of the primers comprising the sequence of SEQ ID NO:4 or SEQ ID NO:6, and/or SEQ ID NO: It can hybridize with two probes.

Methods Provided herein are methods of using the compositions disclosed herein, eg, to monitor the efficiency of NAT assay performance. In some embodiments, the compositions disclosed herein can be used to monitor test sample preparation and processing efficiency. For example, in some embodiments, the compositions provided herein can be combined with a test sample and used to monitor nucleic acid extraction and/or processing (eg, amplification and/or detection). .. In some embodiments, the compositions provided herein are not combined with a test sample, but are processed in parallel to monitor nucleic acid extraction and/or processing (eg, amplification and/or detection). It In some embodiments, the primers and nucleic acid templates disclosed herein serve as a control for amplification of various analytes while still providing extraction efficiency or other sample processing systems for various automated or manual processes. Provide a robust and reproducible method for determining performance metrics for For example, the compositions disclosed herein include, but are not limited to, devices that automatically prepare and process samples in NAT assays (eg, automatically perform nucleic acid extraction and preparation, and amplification and detection). Advantageously, it can be used with devices, including, those that perform NAT assays automatically.

In some embodiments, extraction performance controls are provided. Nucleic acid can be analyzed using a variety of techniques known in the art, such as biological or clinical specimens, such as tissue samples, body fluids, or cell culture samples, and various types of specimens including food samples, soil samples, and the like. Can be extracted from. Nucleic acid extraction can include separating nucleic acids from biological samples, such as proteins, lipids, membranes, organelles, carbohydrates, and other substances in inorganic molecules. Nucleic acid extraction can be performed manually or by one of a variety of automated systems. The methods provided herein provide for, for example, validating extraction methods, devices, and/or reagents to monitor the efficiency of nucleic acid extraction, provide a positive control when the extraction process is performed, and It may be useful to perform maintenance on one or more components of an automated processing device.

In some embodiments, a known amount of nucleic acid template, eg, target control sequence (TCS), is used in a biological sample, eg, SEQ ID NOs: 1, 8, 9, 10, 11, 12, 13, variants thereof, or variants thereof. Added to the reverse complement. In some embodiments, a known amount of nucleic acid template is added to the test sample prior to performing any steps of the nucleic acid extraction protocol. In some embodiments, a known amount of nucleic acid template is added after at least one step in the nucleic acid extraction protocol has been performed.

In some embodiments, the compositions disclosed herein can be used in nucleic acid amplification methods. In some embodiments, nucleic acid amplification can include quantitative nucleic acid amplification, eg, to determine the relative or absolute amount of nucleic acid present in a sample. In some embodiments, the nucleic acid can include qualitative nucleic acid amplification, eg, to determine if the nucleic acid sequence is present or absent in the sample. In some embodiments, nucleic acid amplification, for example, to determine whether a nucleic acid sequence is present in a sample and, if present, to measure the relative or absolute amount of the nucleic acid sequence present in the sample. It may include quantitative and qualitative nucleic acid amplification. Methods of nucleic acid amplification include, but are not limited to, polymerase chain reaction (PCR), strand displacement amplification (SDA), such as multiple displacement amplification (MDA), loop-mediated isothermal amplification (LAMP), ligase chain reaction (LCR), Various transcription-based amplification procedures, including immuno-amplification and transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), self-sustaining sequence replication (3SR), and rolling circle amplification can be mentioned. For example, Mullis, "Process for Amplifying, Detecting, and/or Cloning Nucleic Acid Sequences", U.S. Pat. No. 4,683,195; Walker, "Strand Displacement Amplification 5,5, 16; U.S. Pat. , "Multiple displacement amplification", U.S. Patent No. 6,977,148; Notomi et al., "Process for Synthesizing Nucleic Acid", U.S. Patent No. 6,410,278; Landegren et al., U.S. Patent No. 4,988. , "Method of detecting a nucleotide change in nucleic acids"; Birkenmeyer, "Amplification of Target Nucleic Acids Using Gap Filling Ligase Chain Reaction", US Patent No. 5,427,930; Cashman, "Blocked-Polymerase Polynucleotide Immunoassay Method and Kit U.S. Pat. No. 5,849,478; Kasian et al., "Nucleic Acid Sequence Amplification Methods", U.S. Pat. No. 5,399,491; Malek et al. , 238; Lizardi et al., BioTechnology, 6: 1197 (1988); Lizardi et al., U.S. Pat. No. 5,854,033, "Rolling circle replication reporter systems". In some embodiments, two or more of the listed nucleic acid amplification methods are performed, eg, sequentially.

In some embodiments, a series of individual amplification reactions are performed with known amounts of nucleic acid template, eg, template control sequences (TCS) disclosed herein, to generate a standard curve. Based on the standard curve, the amount of target template in a separate amplification reaction can be determined. In some embodiments, a known amount of an internal control template (eg, a template control sequence such as SEQ ID NO: 1, 8, 9, 10, 11, 12, or 13, or a variant thereof) will increase the target nucleic acid to be amplified. In combination with the sample, which may or may not contain, both the template control sequence and the target nucleic acid (if present) are amplified in multiplex reactions. In some embodiments, each reaction is a series of nucleic acid amplification reactions with a known amount of template control sequence (eg, SEQ ID NO: 1, 8, 9, 10, 11, 12, or 13), the detection threshold is an internal control. A determination is made for each quantity of template, which produces a calibration curve. In some embodiments, multiple replications of each amount of control template are performed, eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10 replications. The standard control sequence calibration curve can then be used to determine the amount of target reactant in the sample. Multiplexed and standard curve methods of nucleic acid quantification are described in detail by McMillan et al. (US Pat. No. 6,783,934). In some embodiments, the number of amplification cycles performed until the target amplicon reaches the detection threshold is calculated (“Cq”). In some embodiments, the method comprises providing a probe during the amplification reaction. For example, in some embodiments, non-sequence specific probes are provided. In some embodiments, the methods disclosed herein provide a probe that comprises, consists of, or consists essentially of a sequence-specific probe, such as SEQ ID NO:2, or a variant thereof, in an amplification reaction. Including steps.

In some embodiments, the method comprises detecting the amount of amplicon produced. The detection can be performed continuously or periodically. For example, the detection can be performed at the end of each Nth cycle or fractions thereof, where N is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 and the like. In some embodiments, the detection measures fluorescence, eg, the intensity of electromagnetic radiation in a wavelength range that includes the emission wavelength of a fluorophore tethered to a probe, or the emission wavelength of a fluorophore tethered to a probe. May be included. In some embodiments, detecting can include detecting FRET.

In some embodiments, the efficiency of quantitative nucleic acid amplification is measured. This measures the efficiency of a quantitative nucleic acid amplification reaction, eg, determines the sensitivity of the reaction to low copy number nucleic acids, or optimizes or validates protocols, reagents, and/or devices for quantitative nucleic acid amplification. Can be useful to do. In some embodiments, a known amount of nucleic acid template is added to the quantitative nucleic acid reaction. In some embodiments, the template is an internal control template. In some embodiments, a primer pair configured to amplify a nucleic acid template amplicon is added to the reaction. The forward and reverse primers of the primer pair can hybridize to the template and extend, thus producing an amplicon. The amount of amplicon produced can be monitored at one or more time points during the amplification reaction, or can be monitored continuously using the methods disclosed herein. In some embodiments, the threshold cycle Cq is a level of relative fluorescence units (RFU), eg, about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, It can be defined as 1400, 1500, 1700, 2000, 2500, or 3000 RFU. In Example 2 herein, Cq is defined as 500 RFU. In some embodiments, the calibration curve is expressed as Cq vs. starting amount of template, or logarithmic transformation thereof. In some embodiments, the calibration curve is expressed as relative fluorescence units versus starting amount of template, or logarithmic transformation thereof.

In some embodiments, a known amount of nucleic acid template is quantified by a quantitative nucleic acid amplification reaction described herein, eg, quantitative PCR. In some embodiments, for example, in the embodiment where RNA is extracted, RNA is reverse transcribed into DNA prior to the quantification step. In some embodiments, a known amount of nucleic acid template is quantified after the extraction procedure is complete. In some embodiments, a known amount of nucleic acid template is quantified after an intermediate step in the extraction process, eg, to determine the efficiency of subsequent steps in the extraction process.

One of ordinary skill in the art will appreciate that the compositions disclosed herein can be used in various types of nucleic acid amplification reactions, as disclosed herein. In some embodiments, the compositions disclosed herein can be used in the polymerase chain reaction (PCR). For a review of PCR techniques, including standard PCR conditions, applied to clinical microbiology, see Dredrecht; Boston: Kluwer Academic, (2000), DNA Methods in Clinical Microbiology, Singleton P. , Methods in Molecular Biology, 67: Human Closing to Genetic Engineering, White, B. in Humana Press, Totowa (1997). A. See eds. and "PCR Methods and Applications," 1991-1995 (Cold Spring Harbor Laboratory Press). Non-limiting examples of "PCR conditions" include the conditions disclosed in the references cited herein, such as 50 mM KCl, 10 mM Tris-HCl (pH 9.0), with an annealing temperature of 72°C. 4mM of MgCl ₂ using 0.1% Triton X-100,2.5mM of MgCl ₂ or annealing temperature of 59 ° C.,, 100 mM of Tris, the pH8.3,10mM KCl, of 5mM _{(NH 4) 2} 50 mM KCl with SO ₄ , 0.15 mg BSA, 4% trehalose, or an annealing temperature of 55° C., 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2. For example, 5 mM MgCl ₂ .

In some embodiments, at least one polymerase is provided. The polymerase can be used for quantitative PCR. FASTSTART™ Taq DNA Polymerase (Roche), KlenTaq 1 (AB peptides Inc.), HOTGOLDSTAR™ DNA Polymerase (Eurogentec), KAPATAQ™ HotStart DNATapA™, or KARTATAQ™ HotStart DNAS™. A variety of nucleic acid polymerases are available for use, including DNA polymerase (Kapa Biosystems), and PHUSION™ Hot Start (Finnzymes).

In some embodiments, a single primer is used for amplification, eg, SEQ ID NOs: 3, 4, 5, 6, or variants thereof.

Temperature Cycling Temperature cycling conditions can vary in time and temperature for each of the different steps depending on the thermal cycler used and other variables that can modify the performance of the amplification. In some embodiments, a two-step protocol is performed in which the protocol combines annealing and elongation steps at typical temperatures that are optimal for both primer and probe annealing and extension steps. In some embodiments, a 3-step protocol is performed in which denaturation, annealing, and elongation steps are performed.

In some embodiments, the compositions disclosed herein provide devices for real-time amplification reactions, such as BD MAX® (Becton Dickinson and Co., Franklin Lakes, NJ), VIPER® ( Becton Dickinson and Co., Franklin Lakes, NJ), VIPER LT (registered trademark) (Becton Dickinson and Co., Franklin Lakes 700, NJ), SMARTCYLCER (registered trademark), Cefunid, Snepheid, Snepheid. (Applied Biosystems, Foster City, CA), ROTOR-GENE(trademark) (Corbett Research, Sydney, Australia), LIGHTCYCLER(R) (Roche Diagnosticacorp. , CA), IMX4000(R) (Stratagene, La Jolla, CA), CFX96(TM) Real-Time PCR System (Bio-Rad Laboratories Inc), and the like.

Isothermal Amplification In some embodiments, the compositions disclosed herein can be used in methods involving isothermal amplification of nucleic acids. Isothermal amplification conditions can vary in time and temperature depending on variables such as the method used, enzyme, template, and one or more primers. Examples of amplification methods that may be performed under isothermal conditions include, but are not limited to, several versions such as LAMP, SDA, etc.

Isothermal amplification can include an optional denaturation step, followed by an isothermal incubation in which the nucleic acids are amplified. In some embodiments, the isothermal incubation is performed without an initial denaturation step. In some embodiments, the isothermal incubation is at least about 25° C., eg, about 25° C., 26, 27, 28, 29, 30, 31, 32, including ranges between any of the listed values. 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, Performed at 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75°C. In some embodiments, the isothermal incubation is performed at about 37°C. In some embodiments, the isothermal incubation is performed at about 64°C. In some embodiments, the isothermal incubation is 180 minutes or less, eg, about 180, 165, 150, 135, 120, 105, 90, 75, including ranges between any two of the recited values. It is carried out for 60, 45, 30, or 15 minutes.

In Situ Hybridization In some embodiments, methods of in situ hybridization are provided. In situ hybridization can be performed on the samples described herein, or sections thereof. In some embodiments, in situ hybridization is used to identify the localization of nucleic acid target sequences, eg, within a cell, within a population of cells, within a tissue or organ, and/or within an entire organism. be able to. In some embodiments, in situ hybridization is used to quantify the copy number of the target nucleic acid in the sample. In some embodiments, the probes described herein can be used for in situ hybridization. In some embodiments, the probe can comprise an oligonucleotide consisting essentially of SEQ ID NO:2, or a variant and/or subsequence thereof. In some embodiments, the probe can comprise an oligonucleotide comprising, or consisting essentially of, one of SEQ ID NOs: 3-6, or a variant and/or subsequence thereof.

Master Mix In some embodiments, a master mix is provided. The master mix may include at least two reagents for the assay provided in relative concentrations that are proportional to the relative concentrations of the reagents in the NAT assay. Therefore, a single amount of master mix can be added to the reaction to provide the appropriate relative concentrations of two or more reagents. In some embodiments, the master mix can include at least two of a polymerase, a buffer, a salt such as magnesium, a nucleotide triphosphate, a primer pair, and water. In some embodiments, the master mix may be provided at a higher concentration than will be used in the reaction. In some embodiments, the master mix is provided in lyophilized form and reconstituted at a higher concentration than will be used in the reaction. In some embodiments, the master mix is at least about 2 times the reaction concentration, eg, 2 times, 2.5 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, It includes reagents at 10-fold, 15-fold, 20-fold, 25-fold, 40-fold, 50-fold, 100-fold, 200-fold, 250-fold, or 500-fold concentrations.

Kits In some embodiments, kits are provided. In some embodiments, the kit comprises a primer pair, one probe, template polynucleotide, eg, a quantitative NAT internal control template sequence, polymerase, buffer, one or more nucleotides (eg, dNTP mix), instructions. Includes one or more of the written or packaging. In some embodiments, at least one reagent of the kit is provided in a master mix.

In some embodiments, the internal control template, at least one primer pair for amplifying the internal control template, and at least one probe hybridized to the amplicon of the primer pair comprise at least one of the target polynucleotides. Of primer pairs and at least one probe.

In some embodiments, the internal control sequence of the kit comprises at least one of SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. Contains a polynucleotide template.

In some embodiments, the kit comprises at least one primer described herein, eg, an oligonucleotide comprising one of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6. Including. In some embodiments, the kit comprises at least one primer pair described herein, eg, primer pair 1, and/or primer pair 5.

In some embodiments, the kit comprises at least one probe described herein, eg, an oligonucleotide probe comprising SEQ ID NO:2 or 16.

In some embodiments, the kit comprises at least one of reverse transcriptase; RNAse, such as RNAase H; or RNA polymerase, such as T7 RNA polymerase.

Quantitative PCR with sensitivity, robustness, and reproducibility
A series of polymerase chain reaction amplification reactions were performed to assess the sensitivity, robustness, and reproducibility of the compositions disclosed herein as control/standard sequences.

Each reaction consisted of a known number of copies of the template plasmid, a TAQMAN™ (Life Technologies) probe having the sequence of SEQ ID NO:2 (or SEQ ID NO:16 for the assay shown in FIG. 4D), and primer pair 1 or primer. It contained one of pair 5. In the assay depicted in Figures 3A, 3B, 4A, 4B, 4C, and 4D, the template was a plasmid having the sequence of SEQ ID NO:8. In the assay depicted in Figure 5, the template was a linearized plasmid having the sequence of SEQ ID NO:15. Reactions were performed with 5 copies of template, 50 copies of template, and 500 copies of template with 4 replicates. Real-time PCR was performed in optical 96-well reaction plates using the CFX96™ Real-Time PCR System (Bio-Rad Laboratories Inc,). The temperature cycling profile was 95°C, 15 minutes (1 cycle); 95°C, 15 seconds, 60°C, 1 minute (50 cycles). PCR reaction conditions were Tris pH 8.0 (70 mM), NaOH 5.0 mM, reverse primer 0.60 μM, forward primer 0.60 μM, Dros. MAX probe (0.40 μM) (Note: 4 different probe lots described herein were used), MgCl ₂ (3.5 mM), dATP (0.05 mM), dCTP (0.05 mM), dGTP ( 0.05 mM), dTTP (0.05 mM) and HGS polymerase (2.7 units). For each amount of template, the amount of product at the end of each cycle was measured in relative fluorescence units and the number of cycles (Cq) required to detect a threshold level of product (measured by fluorescent probe hybridization). The case) was determined. The threshold level for Cq was set at 500 RFU.

Figures 3A, 3B, 4A, 4B, 4C, and 4D illustrate the results of these assays. Highly reproducible results were obtained, even for reactions with only 5 copies of plasmid. As shown in FIG. 3A, the R ² value of the calibration curve plotting the copy number of the template against each Cq of replication was 0.993.

For quantitative PCR reactions with 200, 50, 25, and 5 copies of plasmid per PCR reaction, the amplification curves shown in FIGS. 4A, 4C, and 4D show high sensitivity and reproducibility for primer pair 5. As demonstrated, Figure 4B demonstrates high sensitivity and reproducibility for primer pair 1. FIGS. 4A, 4B, and 4C show that each assay shows the sequence of SEQ ID NO: 2 (ie, of the probe from the different probe lots (FIG. 4A=Lot 1; FIG. 4B=Lot 2, FIG. 4C=Lot 3)). Figure 4D depicts the results using a probe with a single "T" at the 5'end, while Figure 4D shows a probe with the sequence SEQ ID NO: 16 (ie, the "TT" doublet at the 5'end of the probe). Represents the results from the assay used. The results in Figure 4D show that this system even when a variant probe is used (because the T on the 5'end of the probe corresponds to a "C" on the equivalent strand of SEQ ID NO: 16, Note that it does not completely complement the probe binding sequence of the template of SEQ ID NO:8), and has high sensitivity and reproducibility. As shown in FIG. 4B, the detection thresholds and amplifications were determined for reactions with 5 copies of template (reference number 1), 50 copies of template (reference number 2), and 500 copies of template (reference number 3). It was reproducible and had good sensitivity.

Quantitative Amplification Reaction Collect cell samples. RNA is isolated from the cell sample. The isolated sample RNA is reverse transcribed, thus producing a sample cDNA. The lyophilized master mix containing polymerase, buffer, magnesium, and nucleotide triphosphates is reconstituted to double concentration by adding diluent. Equal aliquots of the master mix, as well as 2x sample cDNA (or negative or positive controls); primers for amplifying viral RNA markers, SCORPION™ probes for measuring the amount of amplified viral markers, And the liquid containing the internal control nucleic acid are combined in a thin-walled plastic assay tube.

An internal control is present in each assay tube to monitor the integrity of the PCR reagents and detect the presence of PCR inhibitors. Two external controls are also run. One negative control, containing all reagents but no template DNA, and one positive control, a sample known to contain the target gene, was processed as a sample but served as a control. To run. All controls are run simultaneously with the same reagents in the same amplification reaction. An internal control is performed using a template containing a plasmid having the nucleic acid sequence of SEQ ID NO:8. The internal control comprises a primer pair comprising a first oligonucleotide primer having the sequence of SEQ ID NO:3 and a second oligonucleotide primer having the sequence of SEQ ID NO:4. The primer pair amplifies the partial sequence of SEQ ID NO:6 and produces a polynucleotide product having the sequence of SEQ ID NO:7. The internal control comprises the SCORPION™ probe of sequence SEQ ID NO:2.

The reaction mixture comprises a template nucleic acid, an oligonucleotide primer at a concentration of about 0.4 μM each, a probe at about 0.35 μM, a buffer suitable for the enzyme, salts (in this case MgCl ₂ ), and of course about 0.06 U/ It is a classical combination of polymerase enzymes at the lowest concentration of reaction. The FastStart Taq DNA polymerase enzyme is used as the polymerase. Deoxyribonucleotide triphosphate (dNTP) at a concentration of about 0.15 mM for each (dTTP, dATP, dGTP, and dCTP), and about 0.15 mg/mL bovine serum albumin (BSA) are also added to the reaction mixture. BSA is optional and can help to carry out the reaction even in the presence of PCR inhibitors.

Cycling conditions that allow primer extension and amplification of target DNA include denaturation steps, annealing steps, and polymerization steps. The first step is an initial denaturation step of 15 minutes at 95°C. This is followed by a short denaturation step at 95°C for 1 second, an annealing step at 60°C for 9 seconds, and an elongation step at 72°C for 10 seconds. This cycle is repeated 45 times. There is also a final 10 minute final elongation step at 72°C to ensure that the enzyme has completed extending all single-stranded DNA.

After each cycle of amplification, the amount of internal control polynucleotide product is measured by the intensity (in relative fluorescence units) of the fluorescence intensity emitted by the fluorophore of the internal control SCORPION™ probe.

Claims (14)

An isolated nucleic acid comprising a target control sequence that is at least 97% identical to SEQ ID NO:1;
A forward primer comprising an oligonucleotide that is at least 85% identical to at least one of SEQ ID NO:3 or SEQ ID NO:5;
A reverse primer comprising an oligonucleotide that is at least 85% identical to at least one of SEQ ID NO:4 or SEQ ID NO:6;
An oligonucleotide probe comprising an attached detectable moiety and a sequence that is at least 95% identical to SEQ ID NO:2; and
Specimen, comprising a composition for the internal control in the detection and / or quantification of nucleic acids. The composition of claim 1, wherein the forward primer oligonucleotide is at least 95% identical to SEQ ID NO:3. 3. The composition of claim 1 or 2, wherein the reverse primer oligonucleotide is at least 95% identical to SEQ ID NO:4. 2. The composition of claim 1, wherein the forward primer oligonucleotide comprises SEQ ID NO:3 and the reverse primer oligonucleotide comprises SEQ ID NO:4. 2. The composition of claim 1, wherein the forward primer oligonucleotide comprises SEQ ID NO:5 and the reverse primer oligonucleotide comprises SEQ ID NO:6. 6. The composition of any one of claims 1-5, wherein the oligonucleotide probe comprises SEQ ID NO:2. 7. The composition of any one of claims 1-6, wherein the isolated nucleic acid comprises SEQ ID NO:1. A method for quantifying the amount of nucleic acid in a sample, comprising:
Combining with a sample a polynucleotide comprising a sequence that is at least about 97% identical to the sequence of SEQ ID NO:1.
Contacting the polynucleotide with a forward primer and a reverse primer, the forward primer comprising an oligonucleotide at least 85% identical to at least one of SEQ ID NO: 3 or 5. Comprises an oligonucleotide that is at least 85% identical to at least one of SEQ ID NO: 4 or 6, and the contacting step is performed under standard PCR conditions,
Extending the forward and reverse primers, thereby generating at least one target amplicon;
Contacting an oligonucleotide probe with said at least one target amplicon, said oligonucleotide probe comprising a sequence that is at least 95% identical to SEQ ID NO:2 and further comprising a detectable moiety.
Detecting a signal proportional to the amount of said at least one target amplicon. 9. The method of claim 8, wherein the forward primer oligonucleotide is at least 95% identical to SEQ ID NO:3. 10. The method of claim 8 or 9 , wherein the oligonucleotide of the reverse primer is at least 95% identical to SEQ ID NO:4. 9. The method of claim 8, wherein the forward primer oligonucleotide comprises SEQ ID NO:3 and the reverse primer oligonucleotide comprises SEQ ID NO:4. Wherein said polynucleotide comprises SEQ ID NO: 1, The method according to any one of claims 8-10. The method according to any one of claims 8 to 12 , wherein the oligonucleotide probe comprises SEQ ID NO:2. The method according to any one of claims 8 to 14 , further comprising a step of extracting nucleic acid from the sample, wherein the polynucleotide is combined with the sample before the extraction.

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